Techniques to measure body composition are numerous and include measurements based on: total body water, total body potassium, urinary creatinine excretion, underwater weighing (Goldman, R. F. and Buskirk, E. R., "Body volume measurement by underwater weighing: description of a method", In: Techniques for Measuring Body Composition, J. Brozek (ed.), Washington, D.C.: Nat'l Academy of Sciences (1961), 78-79; Lohman, T. G. et al., "Bone and mineral measurements and their relation to body density in children, youth and adults," Hum. Biol. (1984) 56:667-679), and neutron activation analysis conductivity and bioelectrical impedance (Wolfe, R., "Radioactive and stable isotopes tracers in biomedicine: Principles and practice of kinetic analysis," New York: Wiley-Liss (1992) p. 145-165). 0f the current methods available to measure body composition, estimates are made of fat and fat free mass (FFM). None of the current body composition methods estimate muscle mass directly. All the methods relate back to underwater weighing which is in turn related to previous limited dissection of muscle in human cadavers. Also, such tests only estimate lean tissue, which includes bone, liver and skin.
There is great interest in the athletic population in accurately knowing muscle mass. There is also a need for an accurate method for measurement of muscle mass in order to monitor medical conditions such as nutritional deficiencies and wasting diseases.
There have been many models proposed to measure in vivo amino acid/protein kinetics. Associated with each model are both theoretical and practical problems that must be addressed. Noncompartmental models are widely used but are limited greatly by the necessary assumption that production does not occur in the sampling compartment into which the tracer is administered (Cobelli, C. and G. Toffolo. Compartmental versus noncompartmental modeling for two accessible pools. Am. J. Physiol. (Endocrinol. Metab.) 247: R488-R496, 1984). In contrast, compartmental modeling requires certain physiological assumptions, generally assigning compartments to model various components of a metabolic system. The model is constructed from an understanding of the metabolic system under study and is developed based on relationships between mathematical functions describing the isotopic decay curve and the metabolic system (Wolfe, R., "Radioactive and stable isotopes tracers in biomedicine: Principles and practice of kinetic analysis," New York:Wiley-Liss (1992) 145-165).
3-Methylhistidine has been used as a noninvasive marker of muscle proteolysis in vivo (Young, V. R. and H. N. Munro, "Nt-Methylhistidine (3-methylhistidine) and muscle protein turnover: an overview," Fed. Proc. (1978) 37:2291-2300). The primary sequence of actin and myosin white fibers in skeletal muscle contain the unique amino acid 3-methylhistidine (Johnson, P. et al., "3-Methylhistidine in actin and other muscle proteins," Biochem. J. (1967) 105:361-370). Following degradation of muscle proteins, free 3-methylhistidine is released. Yet, 3-methylhistidine is not reutilized for protein synthesis because it does not have a specific tRNA (Young, V. R. et al, "Metabolism of administered 3-methylhistidine: Lack of muscle transfer ribonucleic acid charging and quantitative excretion as 3-methylhistidine and its N-acetyl derivative," J. Biol. Chem. (1972) 217:3592-3600). 3-Methylhistidine is quantitatively excreted in the urine of man, rat, cattle and rabbit (Long, C. L. et al., "Metabolism of 3-methylhistidine in man," Metabolism (1975) 24:929-935; Young, V. R. et al., "Metabolism of administered 3-methylhistidine: Lack of muscle transfer ribonucleic acid charging and quantitative excretion as 3-methylhistidine and its N-acetyl derivative," J. Biol. Chem. (1972) 217:3592-3600; Harris, C. I. and Milne, G., "The urinary excretion of N-tau-methyl histidine by cattle: validation as an index of muscle protein breakdown," Br. J. Nutr. (1981) 45:411-422; Harris, C. I. et al., "3-Methylhistidine as a measure of skeletal-muscle protein catabolism in the adult New Zealand white rabbit," Biochem. Soc. Trans. (1977) 5:706-708). Therefore, it is thought to be a marker of skeletal muscle protein breakdown.
Urine 3-methylhistidine estimation of muscle proteolysis depends on quantitative collection and accurate measurement of urinary 3-methylhistidine. It is assumed that no metabolism of 3-methylhistidine occurs in vivo, which is true in most species (Harris, C. I. and Milne, G., "The identification of the N-methyl histidine-containing dipeptide, balenine, in muscle extracts from various mammals and the chicken," Comp. Biochem. Physiol. (1987) 86B(2):273-279). However, in the rat 3-methylhistidine is transported to liver and is acetylated. The N-acetyl-3-methylhistidine is the major form excreted in the rat (Young, V. R. et al., "Metabolism of administered 3-methylhistidine: Lack of muscle transfer ribonucleic acid charging and quantitative excretion as 3-methylhistidine and its N-acetyl derivative," J. Biol. Chem. (1972) 217:3592-3600), whereas in the adult human, N-acetyl-3-methyl-histidine accounts for less than 5% of the daily 3-methylhistidine excreted (Long, C. L. et al., "Metabolism of 3-methylhistidine in man," Metabolism (1975) 24:929-935). In sheep (Harris, C. I. and G. Milne, "The urinary excretion of Nt-methyl histidine in sheep: an invalid index of muscle protein breakdown," Br.J.Nutr. (1980) 44: 129-140) and pigs (Harris, C. I. and G. Milne, "The inadequacy of urinary (N-tau)-methyl histidine excretion in the pig as a measure of muscle protein breakdown," Br.J.Nutr. (1981) 45:423-429) 3-methylhistidine is not quantitatively excreted in urine but is retained in muscle as a dipeptide balenine (Harris, C. I. and G. Milne, "The identification of the N-methyl histidine-containing dipeptide, balenine, in muscle extracts from various mammals and the chicken," Comp. Biochem. Physiol. (1987) 86B(2):273-279). Hence, urinary 3-methylhistidine excretion cannot be used to estimate muscle protein breakdown in these species.
A compartmental model for swine or sheep must include a compartment for 3-methylhistidine metabolism other than excretion into a urine compartment. Swine excrete less than 2% of 3-methylhistidine from muscle metabolism into the urine with the majority being retained in muscle as the dipeptide balenine. Therefore, swine not only have a large pool of free 3-methylhistidine in muscle but also a large metabolic "sink" of 3-methylhistidine in the form of balenine. Likewise, sheep excrete approximately 15% of 3-methylhistidine in the urine with the remainder being retained in muscle as the dipeptide balenine. Hence a compartmental model describing the metabolism of 3-methylhistidine in these two species must incorporate these metabolic differences as compared to humans, cattle, rats and rabbits. Although there is a substantial body of literature on the metabolism of 3-methylhistidine, few reports have actually measured daily variability of endogenous 3-methylhistidine excretion. Lukaski et al. (Lukaski, H. C. et al., "Relationship between endogenous 3-methylhistidine excretion and body composition," Am. J. Physiol. (Endocrinol. Metab.)(1981) 240(3):E302-E307) reported an intra-individual coefficient of variation of 4.5% (range 2.2 to 7.0%). Similar intra-individual variation (5%) was reported by Sjolin et al. (Sjolin, J. et al., "Urinary excretion of 1-methylhistidine: A qualitative indicator of exogenous 3-methylhistidine and intake of meats from various sources," Metabolism (1987) 36:1175-1184), whereas interindividual variation (20%) is usually of a much higher magnitude even with large and homogeneous subject populations (Sjolin, J. et al., "Urinary excretion of 1-methylhistidine: A qualitative indicator of exogenous 3-methylhistidine and intake of meats from various sources," Metabolism (1987) 36:1175-1184).
A relationship between fat free mass and urinary 3-methylhistidine has been demonstrated in humans (Lukaski, H. C. and Mendez, J., "Relationship between fat-free weight and urinary 3-methylhistidine excretion in man," Metabolism (1980) 29:758-761; Lukaski, H. C. et al., "Relationship between endogenous 3-methylhistidine excretion and body composition," Am. J. Physiol. (Endocrinol. Metab.) (1981) 240(3):E302-E307; Mendez, J. et al., "Fat-free mass as a function of maximal oxygen consumption and 24-hour urinary creatinine, and 3-methylhistidine excretion," Am. J. Clin. Nutr. (1984) 39:710-714). Urinary creatinine has been suggested as an index of muscle mass and strong correlations have been demonstrated between urinary creatinine and 3-methylhistidine excretion (r=0.87, P&lt;0.001) and between urinary creatinine and muscle mass (Lukaski, H.C. et al., "Relationship between endogenous 3-methylhistidine excretion and body composition," Am. J. Physiol. (Endocrinol. Metab.) (1981) 240(3):E302-E307). Previous studies have attempted to validate the quantitative urinary excretion of 3-methylhistidine. .sup.14 C-labeled 3-methylhistidine was injected, but its isotopic dilution was not described (Long, C. L. et al., "Metabolism of 3-methylhistidine in man," Metabolism (1975) 24:929-935).
There is a controversy as to whether urinary 3-methylhistidine is primarily a product of skeletal muscle protein turnover or whether other tissues might contribute a significant amount to the daily production. Haverberg et al. (Haverberg, L. N. et al., "Nt-Methylhistidine content of mixed proteins in various rat tissues," Biochem. Biophys. Acta (1975) 405:67-71) showed that the mixed proteins in all of the organs sampled contained detectable levels of bound 3-methylhistidine. However, when examining each organ as a whole, it was found that skeletal muscle contained the majority (98%) of the total amount. This study did not include an assessment of the amount of 3-methylhistidine in skin and intestine. Intestinal tissue is also a source of 3-methylhistidine production in humans as measured in urine. Brenner, U. et al. (1987), "Der Einfluss des Dunndarms auf den 3-Methylhistidin-staffwechsel des Menschen (The effect of the small intestine on 3-methylhistidine metabolism in the human)", Infusionther. Klin. Ernahr. 14:248-251. Nishizawa et al. (Nishizawa, M. et al., "Fractional catabolic rates of myosin and actin estimated by urinary excretion of N-methyl histidine: the effect of dietary protein level on catabolic rates under conditions of restricted food intake," Br. J. Nutr. (1977) 37:345-353) concluded that the skin and intestine contributed up to 10% of the total body pool of 3-methylhistidine. Comparative turnover studies of 3-methylhistidine-containing proteins in intestine, skin and skeletal muscle suggest that the skin and the intestine contributed 17% of the 3-methylhistidine excreted per day in the urine (Millward, D. J. and P. C. Bates, "3-Methylhistidine turnover in the whole body, and the contribution of skeletal muscle and intestine to urinary 3-methylhistidine excretion in the adult rat," Biochem. J. (1983) 214:607-615). These calculated values are based on the fitting of exponential regression lines to the specific activity of 3-methylhistidine from the various tissues after giving a dose [methyl-.sub.3 H]methionine. The accuracy of this estimate is uncertain because the labeling technique used may be confounded by the reutilization of labeled methionine (Young, V. R. and H. N. Munro, "Nt-Methylhistidine (3-methylhistidine) and muscle protein turnover: an overview," Fed. Proc. (1978) 37:2291-2300). Millward et al. (Millward, D. J. et al., "Quantitative importance of non-skeletal-muscle sources of N-tau-methyl-histidine in urine," Biochem. J. (1980) 190:225-228) have also concluded that skeletal muscle, skin, and gastrointestinal tract contribute only 25, 7 and 10%, respectively, of the 3-methylhistidine excreted in the adult rat, with the remainder being excreted by some unknown organ. These values were based on a measurement of a decay curve of labeled 3-methylhistidine, after an injection of labeled methionine.
In contrast, following a duodenoileostomy in rats which left only 8% to 10% of small gut intact, it was concluded that the small intestine did not make a significant contribution to a 24-hour urinary excretion of 3-methylhistidine (Brenner, U. et al., "The contribution of small gut to the 3-methylhistidine metabolism in the adult rat," Metabolism (1987) 36:416-418). A similar study with short-bowel humans indicated that skeletal muscle was the major source of urinary 3-methylhistidine. In human patients with varying degrees of infection (Sjolin, J. et al., "Exchange of 3-methylhistidine in the splanchnic region in human infection," Am. J. Clin. Nutr. (1989) 50:1407-1414) it was concluded that urinary 3-methylhistidine was a valid marker of myofibrillar protein breakdown because it was correlated with the release of 3-methylhistidine from the leg. Furthermore, it was later shown with additional patients (Sjolin, J. et al., "Total and net muscle protein breakdown in infection determined by amino acid effluxes," Am. J. Physiol. (Endocrinol. Metab.) (1990) 258:E856-E863) that there was a significant linear relationship between the effluxes of tyrosine and phenylalanine and the efflux and urinary excretion of 3-methylhistidine.
A method to describe 3-methylhistidine metabolism in cattle has been described by using a compartmental model (Rathmacher, J. A. et al., "Technical Note: The use of a compartmental model to estimate the de novo production rate of Nt-methylhistidine in cattle," J. Anim. Sci. (1992) 70:2104-2108). The model showed similar results from plasma and urine. The model estimated 3-methylhistidine production. Fractional muscle breakdown was calculated from the production rate by assuming that muscle mass accounted for 33% of total mass of the steers. (Rathmacher, J. A. et al., "Technical Note: The use of a compartmental model to estimate the de novo production rate of Nt-methylhistidine in cattle," J. Anim. Sci. (1992) 70:2104-2108). Cattle quantitatively excrete 3-methylhistidine into the urine, as do humans (Harris, C. I. and Milne, G., "The urinary excretion of N-tau-methyl histidine by cattle: validation as an index of muscle protein breakdown," Br. J. Nutr. (1981) 45:411-422). 3-methylhistidine production can be described by the model.
A three-compartment mathematical model for analysis of plasma measurements of isotopic and natural 3-methylhistidine in lambs has been tested, resulting in the suggestion that pool (compartment) 3 may be a valid indicator of muscle mass in sheep. (Link, G. A. (1991), "A comprehensive approach to describing protein turnover in lambs," Ph.D. thesis, Department of Animal Science, Iowa State University.) However, sheep metabolize 3-methylhistidine differently than humans, i.e., not all of it is excreted in the urine but, as discussed above, part is converted to balenine and stored in the muscles.
A similar kinetic approach can be utilized in sheep and swine (Rathmacher, J. A. et al., "Measurement of 3-methylhistidine production in lambs by using compartmental-kinetic analysis," Br. J. Nutr. (1992) 69:743-755; Rathmacher, J.A. et al., "Estimation of 3-methylhistidine production in swine by compartmental analysis," J. Anim. Sci. (1992) Abstract, 70:194) which, unlike humans, retain 3-methylhistidine in muscle as the dipeptide balenine (Harris, C. I. and Milne, G., "The urinary excretion of Nt-methyl histidine in sheep: an invalid index of muscle protein breakdown," Br. J. Nutr. (1980) 44:129-140; Harris, C. I. and Milne, G., "The inadequacy of urinary (N-tau)-methyl histidine excretion in the pig as a measure of muscle protein breakdown," Br. J. Nutr. (1981) 45:423-429).
Muscle is degraded in response to many metabolic situations including: starvation, infection, surgery, diabetes, nutrition level, hormonal and stress conditions. Scientific advances in these clinical areas are limited due to limitations in methodology available to quantitate myofibrillar proteolysis versus proteolysis of muscle as a whole. The protein reserve of skeletal muscle is composed of two distinct fractions; myofibrillar protein, the structural component, and non-myofibrillar, non-structural component. The myofibrillar protein makes up 60% of the skeletal muscle protein and turnover slower than non-myofibrillar protein (Bates, P. C. et al., "Myofibrillar protein turnover: synthesis of protein-bound 3-methylhistidine, actin, myosin heavy chain and aldolase in rat skeletal muscle in the fed and starved states," Biochem. J. (1983) 214:593-605). There is also evidence to show that myofibrillar and non-myofibrillar are not under the same metabolic control nor degraded by the same mechanism (Lowell, B. B. et al., "Regulation of myofibrillar protein degradation in rat skeletal muscle during brief and prolonged starvation," Metabolism (1986) 35:1121-1127; Lowell, B. B. et al., "Evidence that lysosomes are not involved in the degradation of myofibrillar proteins in rat skeletal muscle," Biochem. J. (1986) 234:237-240; Goodman, M. N., "Differential effects of acute changes in cell Ca2+ concentration on myofibrillar and non-myofibrillar protein breakdown in the rat extensor digitorum longus muscle in vitro: Assessment by production of tyrosine and N-tau-methylhistidine," Biochem. J. (1987) 241:121-127).
An insignificant increase in 3-methylhistidine excretion in humans during exercise and a significant increase during recovery from exercise has been reported, suggesting that exercise does not result in significant depletion of muscle mass. Carraro, F. (1990), "Effect of exercise and recovery on muscle protein synthesis in human subjects," Am. J. Physiol. 259E470-476.
As an alternative to measuring urinary 3-methylhistidine, the difference between plasma 3-methylhistidine measurements of venous and arterial flows in humans has been used to determine 3-methylhistidine afflux in patients with myotonic dystrophy; this difference was found not to correlate with the presence of myotonic dystrophy, suggesting that myofibrillar protein degradation is not increased in myotonic dystrophy.
All publications referred to herein are incorporated in their entirety by reference.
There is thus a need in the art for the measurement of muscle mass in humans which has greater accuracy and is subject to less interference by extraneous factors than prior art tests.